August 2018
EFFECT OF POD STORAGE AND FERMENTATION DURATIONS USING SHALLOW BOX ON THE QUALITY OF
COCOA (Theobroma cacao L.) BEANS
by
KHAIRUL BARIAH SULAIMAN
Thesis submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
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ACKNOWLEDGEMENT
Alhamdulillah, first of all, I would like to express my profound gratitude to Almighty Allah S. W. T. for giving me the capability and strength to complete this study and my shalawat to His righteous messenger, Prophet Muhammad S.A.W
I would like to take this opportunity to express my deepest appreciation and gratitude to my previous supervisor, Associate Professor Dr Tajul A. Yang, for constantly believing in me to finish this study. His invaluable guidance, constructive suggestions and encouraging advice throughout this study will forever leave a feeling of indebtedness that cannot be fully expressed. I am also very grateful to my current supervisor, Assoc. Prof. Dr Fazilah Ariffin for her constructive comments, and willingness to spend her valuable time towards the preparation of this thesis.
My sincere gratitude to the Ministry of Science, Technology and Environment of Malaysia for the financial support on this project through the ScienceFund grant (06-03-13-SF0115). I am also indebted to Malaysian Cocoa Board especially to Dr Sabariah Samsuddin and Dr Alias Awang for scholarship and laboratory facilities, respectively which without their help, the research work would not be possible. My deepest gratitude is also extended to my colleagues and all the staff of CRDC Bagan Datuk Perak especially to Primary Processing Unit, Mr Husin and Mdm Norasah for their continuous assistance to the success of this study. Acknowledgement is also conveyed to all my study-mates (Dr Wahidu, Intan, Hari and Akmal) and the staff of the School of Industrial Technology for their help and cooperation in many ways to complete my graduate study in USM.
Last but not the least, I also wish to express my deepest appreciation to my beloved husband, Cairil Nidzwan, who has always been there and never failed to show
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his ardent love, sacrifices, patience, endless prayers and support. My beloved children (Khairunnisa Izzati, Khairil Rashid Alhafiz, Khairil Rafique Alhalim and Khairil Razique Alhazim), father, stepmother, mother-in-law, aunties, brothers and cousins for their understanding and constant support through the duration of my graduate study.
Special memory and Alfatihah to my late mother, Hajjah Asiah Kasmat, who encourages me to pursue PhD and taught me the meaning of life.
KHAIRUL BARIAH August 2018
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TABLE OF CONTENTS
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iv
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xii
ABSTRAK xiii
ABSTRACT xv
CHAPTER 1 – INTRODUCTION
1.1 General Background 1
1.2 Problem Statement 4
1.3 Objectives 5
1.4 Hypothesis 6
CHAPTER 2 – LITERATURE REVIEW
2.1 Theobroma cocoa L 7
2.1.1 Cocoa Seed 11
2.1.2 Classification of Cocoa Seed 12
2.1.3 Composition of Cocoa Seed 14
2.2 Cocoa Processing 16
2.2.1 Primary Processing 17
2.2.1(a) Pod breaking and seeds preparation 17
2.2.1(b) Fermentation 19
2.2.1(c) Drying 21
2.2.2 Secondary Processing 22
2.2.2(a) Beans cleaning 23
2.2.2(b) Roasting 24
2.2.2(c) Grinding 25
2.2.3 Cocoa-Based Product Development 25
2.3 Biochemical Changes During Fermentation 26
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2.3.1 Pulp Fermentation 26
2.3.2 Cotyledon Fermentation 29
2.4 Factors Affecting Fermentation 32
2.4.1 Microbial Communities 32
2.4.2 Technique of Fermentation 33
2.4.3 Shallow Box 34
2.4.4 Pod Storage 37
2.4.5 Duration of Fermentation 39
2.5 Quality of Dried Cocoa Beans 40
2.5.1 Colour Quality 44
2.5.2 Flavour Quality 45
2.6 Colour Compounds 46
2.7 Flavour Compounds 47
CHAPTER 3 – EFFECTS OF POD STORAGE AND FERMENTATION DURATIONS USING A SHALLOW BOX TECHNIQUE TO PHYSICOCHEMICAL CHANGES IN WET COCOA BEANS
3.1 Introduction 49
3.2 Materials and Methods 51
3.2.1 Cocoa Pods 51
3.2.2 Design of Experiment 52
3.2.3 Pod Storage 52
3.2.4 Cocoa Fermentation 53
3.2.5 Sampling 54
3.2.6 Sample Preparation 54
3.2.6(a) Pulp 55
3.2.6(b) Powder 56
3.2.6(c) Defatted cocoa powder 56
3.2.7 Analyses 56
3.2.7(a) Temperature 56
3.2.7(b) Total soluble solids 57
3.2.7(c) Acidity 57
3.2.7(d) Total polyphenols 58
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3.2.8 Statistical Analysis 60
3.3 Results and Discussion 60
3.3.1 Changes of Temperature 60
3.3.2 Changes of Total Soluble Solids 64
3.3.3 Changes in Acidity 67
3.3.4 Changes of Total Polyphenols 75
3.4 Summary 77
CHAPTER 4 – EFFECTS OF POD STORAGE AND FERMENTATION DURATIONS USING SHALLOW BOX ON THE QUALITY OF MALAYSIAN DRIED COCOA BEANS
4.1 Introduction 79
4.2 Materials and Methods 84
4.2.1 Wet Fermented Cocoa Beans 84
4.2.2 Drying 84
4.2.3 Sampling 84
4.2.4 Sample Preparation 86
4.2.4(a) Powder 86
4.2.4(b) Defatted cocoa powder 87
4.2.5 Degree of Fermentation 87
4.2.6 Acidity 87
4.2.7 Free Fatty Acids 88
4.2.8 Level of Polyphenols and Antioxidants Activities 89
4.2.8(a) Total polyphenols content 89
4.2.8(b) Total proanthocyanidins content 89
4.2.8(b)(i) Sample extracts 89
4.2.8(b)(ii) 4-(Dimethylamino)cinnamaldehyde reagent
90
4.2.8(b)(iii) Catechin standards 90
4.2.8(b)(iv) Spectroscopic analysis 90 4.2.8(c) Ferric reducing antioxidants power test 91 4.2.9 Sensory Analysis (Quantitative Descriptive Analysis-QDA) 92
4.2.9(a) Preparation of cocoa liquor 92
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4.2.9(b) Sensory 92
4.2.10 Statistical Analysis 93
4.3 Results and Discussion 94
4.3.1 Degree of Fermentation 94
4.3.2 Level of Acidity 98
4.3.3 Level of Free Fatty Acids 103
4.3.4 Level of Polyphenols and Antioxidants Activities 106
4.3.4(a) Total polyphenols content 106
4.3.4(b) Total proanthocyanidins content 111
4.3.4(c) Ferric reducing antioxidants power 114
4.3.5 Sensory Profiles 117
4.4 Summary 124
CHAPTER 5 – CHARACTERIZATION ON THE COLOUR OF THE DRIED COCOA BEANS AS AFFECTED BY POD STORAGE AND FERMENTATION USING A SHALLOW BOX
5.1 Introduction 126
5.2 Materials and Methods 128
5.2.1 Cocoa Beans 128
5.2.2 Surface Colour Characteristic 128
5.2.3 Browning Index 129
5.2.4 Isolation of Colour-related Compound 130
5.2.4(a) Sample preparation 130
5.2.4(b) Colour spectrum 131
5.2.4(c) Identification of colour related compound 131
5.2.5 Statistical Analysis 132
5.3 Results and Discussion 132
5.3.1 Surface Colour Characteristic 132
5.3.2 Browning Index 143
5.3.3 Isolation of Colour-related Compound 146
5.3.3(a) Colour spectrum 146
5.3.3(b) Colour-related compound 150
5.4 Summary 151
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CHAPTER 6 – CHARACTERIZATION ON THE VOLATILE FLAVOUR OF THE DRIED COCOA BEANS AS AFFECTED BY POD STORAGE AND FERMENTATION USING A SHALLOW BOX
6.1 Introduction 153
6.2 Materials and Methods 156
6.2.1 Cocoa Beans 156
6.2.2 Sample Preparation 156
6.2.3 Isolation of Volatiles Compounds 156
6.2.4 Data Analysis 157
6.3 Results and Discussion 157
6.3.1 Cocoa Volatile Compounds 157
6.3.2 Esters 160
6.3.3 Acids 166
6.3.4 Aldehydes 169
6.3.5 Ketones and Hydrocarbons 171
6.3.6 Alcohols 177
6.3.7 Pyrazines 180
6.3.8 Alkaloids and others 182
6.4 Summary 188
CHAPTER 7– CONCLUSIONS AND RECOMMENDATION
7.1 Conclusions 189
7.2 Recommendation for Further Research 192
REFERENCES 193
APPENDICES
LIST OF PUBLICATIONS
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LIST OF TABLES
Page
Table 2.1 The composition of cocoa seed 14
Table 2.2 Basic requirement in grading standard of Malaysian cocoa beans
43
Table 2.3 Grading specification of Malaysian cocoa beans 44
Table 3.1 The total polyphenols in wet cocoa nibs 76
Table 4.1 The scores of the flavour attributes among all the dried cocoa beans
119
Table 5.1 Colour-related compounds identified at different fermentation duration
151
Table 6.1 The numbers of chromatographic peaks at the different duration of pods storage and fermentation
158
Table 6.2 The distributions of chemical groups of compounds at the different duration of pods storage and fermentation
159
Table 6.3 The distributions of the ester compounds in dried cocoa beans at the different duration of pods storage and fermentation
161
Table 6.4 The distributions of the acid compounds in dried cocoa beans at different duration of pods storage and fermentation
167
Table 6.5 The distributions of the aldehyde compounds in dried cocoa beans at different duration of pods storage and fermentation 170
Table 6.6 The distributions of the ketone compounds in dried cocoa beans at different duration of pods storage and fermentation 172
Table 6.7 The distributions of the hydrocarbon compounds in dried cocoa beans at different duration of pods storage and fermentation
175
Table 6.8 The distributions of the alcohol compounds in dried cocoa beans at different duration of pods storage and fermentation 178
Table 6.9 The distributions of the pyrazine compounds in dried cocoa beans at different duration of pods storage and fermentation 181
Table 6.10 The distributions of the alkaloid and other compounds in dried cocoa beans at different duration of pods storage and fermentation
183
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LIST OF FIGURES
Page
Figure 2.1 World cocoa production 8
Figure 2.2 Schematic of the cocoa tree 9
Figure 2.3 Cocoa pods developed in clusters on the main stem and branches of the tree
10
Figure 2.4 Cocoa pods 11
Figure 2.5 Structure of the cocoa seed 12
Figure 2.6 Cross-section of cocoa seeds 13
Figure 2.7 Lengthwise cut section of cocoa seed under light microscopy 15
Figure 2.8 Flow-chart summary of the cocoa processing 18
Figure 2.9 Pod breaking practice 20
Figure 2.10 Trend of microbial and metabolites changes during the pulp fermentation
28
Figure 2.11 Shallow box used for cocoa fermentation 35
Figure 2.12 Slit (white line) on the side and bottom of shallow box 36
Figure 2.13 The cocoa beans appearance before mix 38
Figure 2.14 Mixing of fermented cocoa beans 38
Figure 2.15 Appearance of cocoa beans when fermentation should be
ended 41
Figure 2.16 Lengthwise cut of wet fully fermented cocoa beans 41
Figure 2.17 Method of sampling 42
Figure 3.1 Storage pods in the basket were placed under the roof 53
Figure 3.2 Healthy fresh seeds ready for fermentation 53
Figure 3.3 Fermentation was carried out in the shallow box 55
Figure 3.4 The changes of the temperature inside the fermenting mass 61
Figure 3.5 The changes of the total soluble solids in the cocoa pulp 65
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Figure 3.6 The changes of the pH in cocoa pulp 68
Figure 3.7 The changes of the pH in wet cocoa nib 70
Figure 3.8 The changes of the titratable acidity in cocoa pulp 73
Figure 3.9 The changes of the titratable acidity in wet cocoa nib 74
Figure 4.1 Drying of sample using the natural sun drying on the platform 85
Figure 4.2 Measurement of moisture content in dried cocoa beans using
Protimeter 85
Figure 4.3 Quartering tool for sub-sampling 86
Figure 4.4 The surface colour of the cut cocoa bean 88
Figure 4.5 The EB scores for the dried cocoa beans 95
Figure 4.6 The pH values of the dried cocoa beans 99
Figure 4.7 The TA values of the dried cocoa beans 101
Figure 4.8 The FFA percentages of the dried cocoa beans 105
Figure 4.9 The total polyphenols content of the dried cocoa beans 108
Figure 4.10 The total proanthocyanidins content of the dried cocoa beans 113
Figure 4.11 The ferric reducing activities of the dried cocoa beans 116
Figure 5.1 The surface colour of the cut cocoa bean 129
Figure 5.2 The percentages of the slaty beans 133
Figure 5.3 The percentages of the fully purple beans 136
Figure 5.4 The percentages of the purple-brown beans 138
Figure 5.5 The percentages of the fully brown beans 140
Figure 5.6 The value of browning index of all the dried cocoa beans 144
Figure 5.7 The spectral of all the 24 dried cocoa beans extract in methanol containing HCL at the wavelength range between 300 to 800 nm
147
Figure 5.8 The spectral profile of sample extracts in methanol containing HCL at the wavelength range between 500 to 800 nm
148
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LIST OF ABBREVIATIONS
ha Hectare
kg Kilogram
NaOH Sodium hydroxide
mg Miligram
g Gram
ml Mililiter
µL Microliter
HCl Hydrogen chloride
TPTZ 2,4,6,-Tripyridyl-s-triazine
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KESAN TEMPOH PENYIMPANAN BUAH KOKO DAN FERMENTASI MENGGUNAKAN KOTAK CETEK KE ATAS KUALITI BIJI KOKO
(Theobroma cacao L.)
ABSTRAK
Fermentasi adalah penting untuk pembentukan prekursor warna dan perisa di dalam biji koko, namun dipengaruhi oleh pelbagai faktor termasuk amalan lepastuai. Oleh itu, kesan tempoh penyimpanan buah koko dan penapaian menggunakan kotak cetek pada biji koko Malaysia telah dikaji. Perubahan fizikokimia dalam biji koko basah dan kualiti biji koko kering yang dihasilkan telah dinilai menggunakan rekabentuk rawak lengkap 4 x 6 dengan tempoh penyimpanan buah (0, 2, 4 dan 6 hari) dan fermentasi (0, 24, 48, 72, 96 dan 120 jam) sebagai faktor utama. Pencirian biji koko kering yang dihasilkan dianalisa lebih lanjut kepada profil warna dan komponen meruap secara spektrofotometri, kromatografi cecair prestasi tinggi-pengesan gelombang berubah dan mikroektraksi fasa pepejal-kromatografi gas jisim spektrometri. Hasilnya menunjukkan bahawa penyimpanan buah koko selama empat dan enam hari mempengaruhi kebanyakan perubahan fizikokimia secara signifikan (p < 0.05) terutamanya suhu timbunan yang meningkat melebihi 42 °C dalam masa 24 jam fermentasi berbanding buah tanpa penyimpanan atau penyimpanan selama dua hari. Kualiti biji koko kering yang dihasilkan lebih baik dengan biji koko dari penyimpanan buah selama empat dan enam hari masing-masing mencapai skor peratusan setara coklat penuh 76.2% dan 68.9% yang menunjukkan tahap fermentasi baik seawal 24 jam berbanding 48 jam bagi buah tanpa penyimpanan (65.5%) dan penyimpanan selama dua hari (78.6%).
Keasidan dalam biji koko kering dipertingkatkan dengan nilaian pH antara 5.09 -
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5.53. Selain itu, asid lemak bebas (0.61 hingga 1.22%) di dalam biji koko kering yang dihasilkan adalah lebih rendah daripada 1.75% setara asid oleik iaitu had maksimum yang dibenarkan oleh Codex Alimentarius. Profil sensori bagi likur dari biji koko yang dikeringkan selepas 24 jam fermentasi dari penyimpanan buah selama empat dan enam hari mempunyai rasa koko yang paling kuat, agak pahit dan astringen dengan sedikit masam serta paling hampir dengan profil likur dari biji koko Ghana. Pencirian warna biji koko menemukan dua puncak spektrum penyerapan dan dikenal pasti sebagai asid klorogenik, asid vanila, katekin, asid kafeik, asid ferulik dan asid protokatekik. Manakala sejumlah 281 komponen meruap telah dikenal pasti dari biji koko kering yang dihasilkan dan telah didominasi oleh sebatian ester. Sebagai kesimpulan, penyimpanan buah selama empat dan enam hari sebelum fermentasi selama 24 jam menggunakan kotak cetek boleh digunakan untuk meningkatkan kualiti biji koko kering yang dihasilkan agar setanding dengan kualiti biji koko Ghana.
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EFFECT OF POD STORAGE AND FERMENTATION DURATIONS USING SHALLOW BOX ON THE QUALITY OF COCOA
(Theobroma cacoa L.) BEANS
ABSTRACT
Fermentation is important for the formation of flavour and colour precursor in the cocoa beans which are influenced by various factors including post-harvest practices. Therefore, the effect of pod storage and fermentation duration using a shallow box on the Malaysian cocoa beans was investigated.
Physicochemical changes in wet cocoa beans and quality of the resulting dried cocoa beans were evaluated using a 4 x 6 complete randomized design with pod storage (0, 2, 4 and 6 days) and fermentation duration (0, 24, 48, 72, 96 and 120 hours) as the principal factors. The resulting dried cocoa beans were further characterized for profiles of colour and volatile compounds using the spectrophotometer, high-performance liquid chromatography-variable wavelength detector and solid-phase microextraction-gas chromatography-mass spectrometry. The results showed that the pod storage for four and six days were significantly (p < 0.05) influenced most of the physicochemical changes especially the mass temperature which had increased exceeding 42 °C within 24 hours of fermentation compared to without storage or pod storage for two days. The quality of dried cocoa beans produced significantly improved with the beans from the pod storage for four and six days had achieved respective scores of equivalent percent of fully brown 76.2% and 68.9% which indicated a well degree of fermentation as early as 24 hours compared to 48 hours from the pods without storage (65.5%) and storage for two (78.6%) days.
The acidity in dried cocoa beans was enhanced with the pH between 5.09 - 5.53.
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Besides, the free fatty acids (0.61 to 1.22%) was lower than the acceptable limit of 1.75% oleic acid equivalent allowed by Codex Alimentarius. Sensory profiles of liquor from the cocoa beans which dried after 24 hours of fermentation from the pod storage for four and six days had the strongest cocoa flavour, moderately bitter and astringent and slightly low in sourness as well as closest to the profile of liquor from the Ghanaian beans. Colour characterization revealed two spectrum peaks and identified as chlorogenic acid, vanillic acid, catechin, caffeic acid, ferulic acid and protocatechuic acid. While a total of 281 volatile compounds identified from the dried cocoa beans, they were dominated by the ester compounds. As conclusions, the pod storage for four and six days prior fermentation for 24 hours using shallow box can be used to enhance the quality of dried cocoa beans produced and comparable to quality of the Ghanaian cocoa beans.
1 CHAPTER 1 INTRODUCTION
1.1 General Background
Theobroma cacao L. is commonly known as cocoa and the seeds which are embedded inside the pod are the only source of raw material for the chocolate and cocoa-based industries (Thompson et al., 2013; Kim et al., 2011).
Therefore, cocoa is recognized as one of the ten most active agricultural commodities being traded in the world (InvestorGuide, 2017; Fry, 2011). In Malaysia, cocoa is the fourth commodity which provides job opportunities for more than 31 thousand people including estate workers, smallholders, cocoa processors, manufacturers as well as chocolate entrepreneurs (Abdel Hameed and Arshad, 2014). The industry has contributed RM5.03 billion of the total value of Malaysia’s major commodities export earnings in 2015 and has been increased to RM5.74 billion in 2016. The increment is driven by increasing exportation of cocoa-based products such cocoa butter and powder (Harnie, 2017; Anon, 2016).
Currently, Malaysia is the 8th largest cocoa grinder in the world, after the Netherlands, Cote d'Ivoire, Indonesia, Germany, United States, Brazil, and Ghana (Harnie, 2017). Despite of the increasing demand of dried cocoa beans for grinding industry, the cultivation area and production of cocoa beans are declining. Since 1996, the smallholder sector has become the dominant players in cocoa cultivation with an average area of 94,716 ha as compared to 73,503 ha under estate sector. To date, the Malaysian cultivation area is about 18,122 ha with 95% (17,243 ha) share of smallholders (Anon, 2015). Changing of a dominant player in the cocoa cultivation has affected the production trend of
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cocoa seeds. In which the estate usually produce as low as 500 kg cocoa seeds per harvesting day while smallholders only produced as high as 20 kg (Anon, 2015).
After harvesting, cocoa seeds have to undergo a series of processing stages starting from fermentation and drying, subsequently followed by roasting, before a unique chocolate flavour is fully developed. Among these processing stages, fermentation is the most crucial because any imperfections in flavour during this stage is irreversible and will affect the taste in final products. Any effort to improve the flavour in the next stage, especially roasting will only increase the cost of operation and will be very expensive (Hidayatullah et al., 2016; De Vuyst and Weckx, 2016; Beckett, 2015b). Generally, cocoa fermentation practices vary considerably from one producing country to another and even from farm to farm within the same region. Approximately half of the world’s cocoa industry is reported to ferment the seeds in boxes while remaining half fermented in heaps, trays or by using other primitive practices such as basket and bucket (Thompson et al., 2013).
Regardless of any fermentation practices have been applied, the process should be conducted properly as the high and consistent quality of dried cocoa beans is one of the main factors in determining the price. Normally, dried cocoa beans come in the form of batch to manufacturer and each of the batch will has some inconsistent qualities especially in flavour. The manufacturers have to put an extra budget to reduce the fluctuation of quality by blending or adding with other substances such as vanilla before the dried cocoa beans are further processed. If the cocoa grinders or chocolate manufacturers get the high and consistent quality of dried cocoa beans, their production costs will be lowered. Hence, the budget that has been saved can be used as an incentive to farmers by offering extra premium price from markets of dried fermented cocoa beans (Abdel Hameed et al., 2009).
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The changes of the players background from estate owners to smallholders in Malaysian cocoa cultivation have not only affected the production trend of cocoa seeds but also the quality of dried cocoa beans. Survey on smallholders from all over the Malaysia has revealed that knowledge level on the importance of fermentation process is still lacking among smallholders. About 59%
of the smallholders have been carried out an improper fermentation process by using various fermentation practices (Albert et al., 2015). Although the Malaysian Cocoa Board (MCB) has recommended the shallow box fermentation as standard practice since 1999, it is not widely used due to the loading capacity. The smallest shallow box needs at least 25 kg cocoa seeds, but the smallholders usually producing less than required capacity per harvesting day. The smallholders tend to conduct in small batches using primitive practices such as rattan basket, plastic bucket, sack, tray and heap (Hii and Bakri, 2002). Therefore, the cocoa beans which have been produced by smallholders were various in qualities and sold at a discounted price in the world market (Hii et al., 2004).
In order to ensure consistency in the quality of the Malaysian dried fermented cocoa beans, the usage of shallow box fermentation is still recommended as it is reported as a well-performed fermentation practice (Papalexandratou et al., 2013; Kelvin et al., 2013; Zaibunnisa, 2002). Modification on the shallow box such as moveable wall or partition is made to cater for smaller cocoa seeds capacity and at the same time ensure that the depth of fermenting mass still 30 cm. The 30 cm depth of fermenting mass is important as Shamsudin et al., (1978) reported that it ensure enough heat generated during fermentation process. The smallholders are also suggested to do pod storage, a practice of storing the cocoa pods for maximum 10 days before fermentation process. The practice is not only helping the farmers to
4
obtain enough cocoa seeds but also can enhance the flavour (Afoakwa et al., 2011a;
Nazaruddin et al., 2006). However, all the modification and suggestion are failed to give consistent quality and often faced the problem of over fermented seeds. On the other hand, the combination of pod storage and shallow box technique is time consuming because the process requires ten days of cocoa storage before five days of fermentation and takes additional three to seven days to allow the cocoa beans completely dry. In sum, the following problems are the issues discussed in order to improvee the quality of Malaysian dried fermented cocoa beans.
1.2 Problem Statements
i. The flavour of Malaysian cocoa beans is inconsistent and often regards as excessive acidic flavour, weak cocoa flavour and also with certain undesirable flavour such as mouldy and hammy. Thus it has been sold at RM5800 to RM 7150/tonne compared to RM10000/tonne of the world market (Harnie, 2017). Cocoa processors normally blend it with higher quality beans; hence increase their processing cost.
ii. The previous researcher Said et al., (1988), has implemented pod storage of ten days prior the fermentation process. However, the implementation of pod storage for ten days prior fermentation is failed to produce consistent good quality beans. This is because the smallholders are not aware of the correct time to terminate the fermentation process. Normally, they just follow the previous five days recommended duration by Malaysian Cocoa Board and faced the over fermentation problem, where the over-fermented cocoa beans are manifested by blackish with mouldy and hammy flavour.
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iii. The quality of cocoa beans produce by smallholders is inconsistent due to the duration of the fermentation is standardized to five day regardless the implementation of pod storage or not. The fermentation duration of five days is shortened from six days duration which is adapted from Ghana and implemented without conducting proper optimization. Therefore, the observations for the best duration of pod storage as well as the fermentation process are needed.
1.3 Objective
The main objective of this study was to determine the effects of the duration of pod storage and fermentation process using the shallow box technique on the quality of Malaysian cocoa beans. The objectives are specifically divided as follows;
i. To evaluate the effects of pod storage duration during fermentation using a shallow box on the physicochemical changes of wet cocoa beans.
ii. To assess the effects of pod storage and fermentation duration using a shallow box on the quality of Malaysian dried cocoa beans and compare its sensory profiles to the Ghanaian dried cocoa beans profiles.
iii. To characterize the colour of the dried cocoa beans as affected by pod storage and fermentation using a shallow box.
iv. To characterize the volatile flavour of the dried cocoa beans as affected by pod storage and fermentation using a shallow box.
6 1.4 Hypothesis
In this study, it is hypothesized that pod storage will reduce the remarkable amount of pulp sugars. The reduction will cause a rapid increase in temperature as well as decreases the formation of acid. At the same time, the usage of the shallow box will provide sufficient heat which helps to enhance the enzymatic reaction and significantly promote faster fermentation. Termination of the fermentation process at the correct time will maximize colour and flavour precursors, hence significantly improve the colour and flavour quality of produced dried cocoa beans.
7 CHAPTER 2 LITERATURE REVIEW
2.1 Theobroma cacao L.
Theobroma cacao L., commonly known as cocoa, is a perennial tree that formerly classified in the family of Sterculiaceae; but later reclassified into the Malvaceae family. The tree is widely cultivated in a range between 20° north and 20°
south of the equator (Figure 2.1a) with three main growing regions, which are West Africa, South-East Asia and South America (Lopes and Pires, 2014; Colombo et al., 2012; Bernaert et al., 2012). Côte d'Ivoire is on the top among the 15 cocoa producing countries with estimated 1581 thousand tonnes of cocoa beans have been produced for 2015/16 crop year (Figure 2.1b) and Malaysia has been in the rank of 23 with production of 1.757 thousand tonnes of cocoa beans (Harnie, 2017). The cocoa tree needs general climatic conditions such as yearly rainfall distribution of 1250 - 3000 mm, preferably between 1,500 - 2000 mm. The minimum means of temperature is in between 19 to 21 °C and maximum temperature 30 to 32 °C with no persistent strong winds as it may damage the plants. In addition, the dry season should be not exceeding three consecutive months with not less than 100 mm rain per month. Besides climatic conditions, the cocoa tree also needs suitable soil properties such as more than 100 mm depth of soil as well as good drainage and water holding capabilities to grow well (Ahmad Kamil et al., 2013).
The cocoa tree can be propagated by two methods, either via seedling or vegetative methods such as cuttings, grafting, and marcotting. The growth of cocoa seedling which known as hybrid differs from the vegetative propagation, where it has one main stem grows vertically upwards until it reaches a certain height
8
Figure 2.1: World cocoa production. (a) Cocoa cultivation regions and (b) Top 15 cocoa producing countries. (Source: Adapted from cocoanibs.wordpress.com and ICCO, 2017). Denotes the cocoa producing country in the world.
Cocoa Producing Country Latin America:
16% (755,000 tonnes) Africa:
76% (3.565 million tonnes )
Asia & Oceania:
8% (379,000 tonnes)
(a)
(b)
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before forming 'jorquette' (Figure 2.2). On top of the 'jorquette' will protrude three to five lateral branching commonly known as fan branches. In contrast, the cocoa tree from vegetative propagation or familiar as clone has only fan branches. On top of the tree morphology, the pod and bean characteristics of the clone are more uniform compared to the hybrid. In addition, certain clones can bear fruit throughout the year and are not depend only on the two peak seasons. Thus, these will allow the farmers to have a stable source of income and due to that, grafting is the highly recommended breeding technique for cocoa in Malaysia (Ahmad Kamil et al., 2013;
Toxopeus, 2008; Francis et al., 2005).
Figure 2.2: Schematic of the cocoa tree. (a) hybrid and (b) clonal (Source: Ahmad Kamil et al., 2013).
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Depending on the propagation method, the tree will start bearing a drupe fruit after 18 - 30 months being planted in the field and remain productive for several decades. The fruit, also known as cocoa pod, develops in clusters from pollinated flowers which arise directly on the auxiliary bud of main stem as well as branches of the tree (Figure 2.3). The pod will take approximately four to six months to ripen and may differ in terms of size, shape, and colour depending on the variety. The surface colour of pod husk which can generally be either green or red when immature, will turn to yellow or orange (Figure 2.4) upon ripening. During harvesting, the pod should be cut on stalk closely to husk using sharp knives, secateurs or machetes to avoid damage to the flower cushions. Damaging the flower cushions or leaving the pod rotten on the tree will reduce the future yields. Breaking the pod will expose about 20–50 of seeds (refer to the fresh and unfermented seeds) which surrounded by a mucilaginous pulp and embedded to a placenta (Zhang and Motilal, 2016; Ahmad Kamil et al., 2013, Thompson et al., 2013).
Figure 2.3. Cocoa pods developed in clusters on the main stem and branches of the tree. (Source: Malaysian Cocoa Board gallery (2016), reprinted with permission.)
(b) (a)
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Figure 2.4. Cocoa pods. (a) Variation of colour and size of cocoa pods (b) Cocoa seeds inside the pod husk. (Source: Personal collection).
2.1.1 Cocoa Seed
Cocoa seed is a fresh bean that serves as the primary source of raw material for the chocolate and cocoa-based industries. The seed structure encompassed of three parts that are mucilaginous pulp, seed coat and cotyledon (Figure 2.5). A white and sweet mucilaginous pulp is the outermost part that surrounding the seed. In which, the pulp or external fermentation of cocoa beans takes place and a decisive factor in the outcome of the fermentation step (Nielsen, 2006). Inside of the seed are the cotyledons as the main part, which consists of two convoluted cotyledons and attached together with small germ or embryo. Seed coat, namely as testa is located between cotyledon and pulp, serves as a barrier to control the diffusion of molecules either going inside or outside of the cotyledon by allowing only smaller molecules such ethanol and acetic acid to diffuse into the cotyledons (Lopes and Pires, 2014;
Ahmad Kamil et al., 2013; Toxopeus, 2008).
(a) (b)
Figure 2.5. Structure of the cocoa seed. (
2.1.2 Classification of Cocoa Seed
Cocoa seed is traditionally classified into three well
namely Forastero, Criollo, and Trinitario. The classification is based on geographical origins and morphological traits such as colour as well as flavour (
2013; Motamayor et al
Amazon region and caters for more than 95% of the world's cocoa supply (Kongor al., 2016; Saltini et al
vigorous and resistant to pests and diseases 2014; Rusconi and Conti, 2010). Cross of cotyledon are in deep purple (Figure
produce strong inherent flavour, incline to be somewhat bitter and usually dark brown in colour (Beck
On the other hand, Criollo is native to Central and South America as well as the Caribbean Islands and Sri Lanka. It accounts for only 5% of the world’s
12
Figure 2.5. Structure of the cocoa seed. (Source: Personal collection
Classification of Cocoa Seed
Cocoa seed is traditionally classified into three well
namely Forastero, Criollo, and Trinitario. The classification is based on geographical origins and morphological traits such as colour as well as flavour (
et al, 2008; Bartley 2005). Forastero is originated from the region and caters for more than 95% of the world's cocoa supply (Kongor
et al., 2013; Lima et al., 2011). The forastero tree
vigorous and resistant to pests and diseases as compared to others (Lopes and Pires, usconi and Conti, 2010). Cross-section of the seeds display
of cotyledon are in deep purple (Figure 2.6a) and upon proper processing will produce strong inherent flavour, incline to be somewhat bitter and usually dark brown in colour (Beckett, 2015a).
On the other hand, Criollo is native to Central and South America as well as the Caribbean Islands and Sri Lanka. It accounts for only 5% of the world’s
Personal collection)
Cocoa seed is traditionally classified into three well-known varieties namely Forastero, Criollo, and Trinitario. The classification is based on geographical origins and morphological traits such as colour as well as flavour (Trognitz et al., , 2008; Bartley 2005). Forastero is originated from the region and caters for more than 95% of the world's cocoa supply (Kongor et ., 2011). The forastero tree variety is more compared to others (Lopes and Pires, section of the seeds display that the surface 2.6a) and upon proper processing will produce strong inherent flavour, incline to be somewhat bitter and usually dark
On the other hand, Criollo is native to Central and South America as well as the Caribbean Islands and Sri Lanka. It accounts for only 5% of the world’s
production (Beckett, 2015a; Jahurul
very pale purple in colour (Figure 2.6b) and have more aromatic or smoother tastes but milder cocoa flavour. The Criollo seed tends to be bigger and rounder as well as having lower fat content
2013). Whereas, Trinitario is reported as the result from hybridization of Forastero and Criollo, which colour characteristic has reflected the variation of Forastero and Criollo (Figure 2.6c) and noted for its fine flavour (Giacometti
Toxopeus, 2008). Besides the three well known as Nacional which
(‘Arriba’) and only grown in Equador (Giacometti However, recently Motamayor
into ten major groups based on genetic differentiation using simple sequence repeat (SSR) analysis: Maran˜on, Curaray, Criollo, Iquitos, Nanay, Contamana, Amelonado, Puru´s, Nacional and Guiana
Figure 2.6: Cross-section of cocoa seeds. (a) Forastero; (b) Criollo and (c) Trinitario.
(Source: Personal collection
13
production (Beckett, 2015a; Jahurul et al., 2013). Criollo seeds are white, ivory or colour (Figure 2.6b) and have more aromatic or smoother tastes but milder cocoa flavour. The Criollo seed tends to be bigger and rounder as well as having lower fat content as compared to Forastero (Beckett, 2015a; Jahurul
hereas, Trinitario is reported as the result from hybridization of Forastero and Criollo, which colour characteristic has reflected the variation of Forastero and Criollo (Figure 2.6c) and noted for its fine flavour (Giacometti
). Besides the three well-known groups, there is another group known as Nacional which has been acknowledged for its raisins fruit
(‘Arriba’) and only grown in Equador (Giacometti et al., 2015; Counet
However, recently Motamayor et al., (2008) has proposed to classify the cocoa tree into ten major groups based on genetic differentiation using simple sequence repeat (SSR) analysis: Maran˜on, Curaray, Criollo, Iquitos, Nanay, Contamana, Amelonado, Puru´s, Nacional and Guiana.
section of cocoa seeds. (a) Forastero; (b) Criollo and (c) Trinitario.
Personal collection)
., 2013). Criollo seeds are white, ivory or colour (Figure 2.6b) and have more aromatic or smoother tastes but milder cocoa flavour. The Criollo seed tends to be bigger and rounder as well as compared to Forastero (Beckett, 2015a; Jahurul et al., hereas, Trinitario is reported as the result from hybridization of Forastero and Criollo, which colour characteristic has reflected the variation of Forastero and Criollo (Figure 2.6c) and noted for its fine flavour (Giacometti et al., 2015;
known groups, there is another group raisins fruit-flavoured ., 2015; Counet et al., 2004).
., (2008) has proposed to classify the cocoa tree into ten major groups based on genetic differentiation using simple sequence repeat (SSR) analysis: Maran˜on, Curaray, Criollo, Iquitos, Nanay, Contamana,
section of cocoa seeds. (a) Forastero; (b) Criollo and (c) Trinitario.
14 2.1.3 Composition of Cocoa Seed
In general, the composition of the cocoa seed is shown in Table 2.1. The cocoa pulp is comprised of spongy parenchyma cells which have wall structure that supported by 1 - 2% pectin (Afoakwa et al., 2013b). The total sugar content including pentosans is approximately 10 - 15%, with the main sugar of the fresh pulp are glucose (5.4 - 6.6%), fructose (6.3 - 7.4%) as well as small amounts of sucrose (less than 0.3%). The ratio of glucose/fructose to sucrose change with the degree of maturity where the pulp in immature cocoa pods consist of a higher proportion of sucrose, while the pulp in ripe cocoa pods containing mainly fructose and glucose.
The pH is relatively low (3.0 - 4.0) mainly due to the content of 0.5 – 3% citric acid (Aprotosoaie et al., 2016; Afoakwa, 2010).
The cocoa cotyledons are composed of two types of parenchyma storage cells, known as lipid/protein cells and polyphenolic cells. The polyphenolic cells
Table 2.1: The composition of cocoa seed.
Component Composition
Pulp
- Water 80 - 87%
- Sugars (glucose, fructose, sucrose) 10 - 13%
- Pentosans 2 - 3%
- Citric acid 0.5 - 3%
- Salts 8 - 10%
Cotyledon
- Water 32 - 39%
- Cellulose 2 - 3%
- Starch 4 - 6%
- Pentosans 4 - 6%
- Sucrose 2 - 3%
- Fat 49.9 - 55.2%
- Proteins 8 - 10%
- Theobromine 2 - 3%
- Caffeine 1%
- Acids 1%
- Polyphenols 5 - 6%
which are dark-stained in Figure 2.7 forming by plasma (Guilherme
cells contain a large number of lipid globules, starch granules as well as protein bodies which are embedded in the cytoplasm. Other organell
mitochondria are also squeezed together among the vacuoles. The polyphenolic cells are larger cells with a large vacuole and contain almost all of the seed's polyphenolic material as well as alkaloids (
al., 2001).
Although lipid/protein cells are smaller than polyphenolic cells, lipid content constitutes about 49.9%
of the fresh cotyledons (Afoakwa
2009). The protein storage in the cotyledons
into four groups albumin, globulin, glutelin and prolamin (Bertazzo The albumin is abundantly present but
cocoa flavour precursor. Only globulin which later known as vicilin (7S) globulin, is found to be significantly degraded during fermentation to produce
Figure 2.7: Lengthwise cut section of cocoa seed under light microscopy de Brito et al., 2001). The dark
15
stained in Figure 2.7 are separated from lipid/protein cells via a grid Guilherme et al., 2016; Martini et al., 2008
cells contain a large number of lipid globules, starch granules as well as protein bodies which are embedded in the cytoplasm. Other organelles such as nuclei and mitochondria are also squeezed together among the vacuoles. The polyphenolic cells are larger cells with a large vacuole and contain almost all of the seed's polyphenolic material as well as alkaloids (Voigt and Lieberei, 2014; Martini et al
Although lipid/protein cells are smaller than polyphenolic cells, lipid content constitutes about 49.9% - 55.2%, while protein composes 17.5%
of the fresh cotyledons (Afoakwa et al., 2013b; Elwers et al., 2010; Belitz storage in the cotyledons is classified based on their solubility into four groups albumin, globulin, glutelin and prolamin (Bertazzo
The albumin is abundantly present but do not contribute in produ
cocoa flavour precursor. Only globulin which later known as vicilin (7S) s found to be significantly degraded during fermentation to produce
Lengthwise cut section of cocoa seed under light microscopy ., 2001). The dark-stained cells are polyphenolic cells.
lipid/protein cells via a grid ., 2008). The lipid/protein cells contain a large number of lipid globules, starch granules as well as protein bodies es such as nuclei and mitochondria are also squeezed together among the vacuoles. The polyphenolic cells are larger cells with a large vacuole and contain almost all of the seed's polyphenolic et al., 2008; de Brito et
Although lipid/protein cells are smaller than polyphenolic cells, lipid 55.2%, while protein composes 17.5% - 21.6%
., 2010; Belitz et al., classified based on their solubility into four groups albumin, globulin, glutelin and prolamin (Bertazzo et al., 2011).
not contribute in producing specific cocoa flavour precursor. Only globulin which later known as vicilin (7S)-class s found to be significantly degraded during fermentation to produce Lengthwise cut section of cocoa seed under light microscopy. (Source:
stained cells are polyphenolic cells.
16
hydrophilic polypeptide and free hydrophobic amino acids: specific cocoa flavour precursors (Guilherme et al., 2016; Voigt and Lieberei, 2014; Marseglia et al., 2014; Kratzer et al., 2009; Amin et al., 2003; Amin et al., 2002).
The polyphenolic cells make up about 12 to 20% of dry weight beans, containing with polyphenols and alkaloids including caffeine, theobromine and theophylline (Aprotosoaie et al., 2016; Voigt and Lieberei, 2014). The polyphenols are comprised of many classes of compounds including flavonoid. The flavonoid is the predominant class in cocoa and further subdivided into three groups which are proanthocyanidins (58%), flavanols (37%) and anthocyanins (4%). The flavanols can occur both as monomers of epicatechin and catechin or polymerized flavanols or procyanidins (Kongor et al., 2016; Bordiga et al., 2015; Voigt and Lieberei, 2014; Ackar et al., 2013; Hurst et al., 2011; Jalil and Amin, 2008). The anthocyanins content is responsible for the fresh cotyledon colour which is being used as an indicator for the degree of fermentation. However, anthocyanins have not been detected in the criollo type beans (Amoa-Awua, 2014; Wollgast and Anklam, 2000; Hansen et al., 2000). The remaining content of the cotyledon are 4 - 6% starch, 2 - 3% cellulose and 1% acids (Voigt and Lieberei, 2014; Nielsen, 2006; Goto et al., 2002; Bucheli et al., 2001).
2.2 Cocoa Processing
Cocoa seeds which are directly dried under the sun are usually lack of chocolate flavour and aroma characteristic. The seeds must go through several stages of processing in order to obtain the desired flavour and colour before being served at the table (Aprotosoaie et al., 2016; Aculey et al., 2010). The process can be classified into three stages, namely primary, secondary and product development.
17
The primary stage is carried out by cocoa farmers at farm involving a series of the process after harvesting including fermentation and drying in order to produce fermented and dried cocoa beans (Fowler, 2017; Kongor et al., 2016). Some farmers in certain countries such as Ghana and Malaysia are practising pod storage prior fermentation either to ensure enough seeds to ferment or to enhance the quality of the dried cocoa beans.
The dried fermented cocoa beans are subsequently been processed into the semi-finished cocoa products such as cocoa butter, cake, powder as well as liquor at the secondary stage by industry through roasting, winnowing, grinding and pressing. Subsequently after that, the semi-finished product will be further transformed at the product development stage into cosmetics, beverages, confectionery, and chocolate for consumer usage (Thompson et al., 2013; Schwan and Wheals, 2004). A flow-chart summarizing the cocoa processing is given in Figure 2.8 (Beckett, 2015b; ICCO, 2014).
2.2.1 Primary Processing
In primary processing, the cocoa seeds will undergo either enzymatic or non-enzymatic transformation to produce flavour precursors in fermented dried cocoa beans. The practices may vary according to the size of the farm and pod yield but the aim is still same, towards high-quality fermented dried cocoa beans (Ahmad Kamil et al., 2013; Amanquah, 2013).
2.2.1(a) Pod breaking and seeds preparation
After been harvested, the pods must be broken to prepare the cocoa seeds for fermentation. The preparation can be carried out either at the farm or other places
18
Figure 2.8: Flow-chart summary of the cocoa processing (Adapted from Beckett 2015b; ICCO, 2014).
PRIMARY PROCESSING
SECONDARY PROCESSING
PRODUCT DEVELOPMENT
Cooling Milling Sieving
Deodourising Cooling Packing
Pod preconditioning Pod breaking Sorting
Cleaning Crushing Winnowing Alkalising (optional)
Blending Mixing Refining Conching Tempering
19
as long as it is near to the processing centre, where fermentation will take place (Lee et al., 2014; Ahmad Kamil et al., 2013). The commonest practice for pod breaking is by manually hitting them together. However, the suggested practice is to use a special knife, which has a ‘block' that will prevent the seeds from getting cut (Figure 2.9a).
Manual pod breaking is considered a time-consuming and labour intensive process.
Hence, applying the mechanical cocoa pod breaker or COBRE™ (Figure 2.9b) is most recommended as extracted seeds should be fermented in the same day after breaking.
The fermentation should be carried out as soon as the seeds have been removed and sorted from placenta or husk fragment. Upon completing the sorting, the seeds will be placed into a container and ready to ferment. Yet, if the process is being carried out in the field, only ready to ferment seeds will be taken to the processing centre, while the husks and pods that have been infested by diseases and insects will be left in the field. This practice is not encouraged because it will spread the diseases and insects at the farm. In contrast, centralized pod breaking at other places near the processing centre will help to control disease and insects spread.
2.2.1(b) Fermentation
Fermentation is identified as a crucial process because involving the enzymatic reaction on carbohydrate, protein, fat and polyphenols. As a result, the cocoa-specific flavour precursors such as reducing sugars, peptides, amino acids (Voigt and Lieberei, 2014, Romero-Cortes, et al., 2013, Khairul Bariah, 2010) as well as key volatile fractions such as alcohols, esters and fatty acids (Aprotosoaie et al., 2016; Rodriguez-Campos et al., 2012) are formed at this stage.
Besides developing the precursors of cocoa flavour and aroma, fermentation also prevents cocoa seeds from germinate and helps to remove the mucilaginous pulp in
Figure 2.9: Pod breaking practice. (a) Manually using special knife and (b) mechanical using
collection and (b) Malaysian Cocoa Board gallery permission.)
20
Figure 2.9: Pod breaking practice. (a) Manually using special knife and (b) mechanical using cocoa pod breaker COBRE™. (Source: (a) Personal collection and (b) Malaysian Cocoa Board gallery (2008), reprinted with Figure 2.9: Pod breaking practice. (a) Manually using special knife and (b) . (Source: (a) Personal (2008), reprinted with
21
order to facilitate drying. Furthermore, the unfermented cocoa seeds do not produce a good cocoa flavour during roasting (Voigt et al., 2016; Schwan and Wheals, 2004).
Fermentation begins as soon as the cocoa seeds are exposed to the environment and microbial is started to grow on the pulp and leads to the production of alcohol and organic acids with concomitant of temperature increment (Schwan et al., 2014). Currently, the succession of microbial during fermentation is well documented involving enterobacteria, indigenous yeasts, lactic acid bacteria (LAB), acetic acid bacteria (AAB), bacilli and filamentous fungi (De Vuyst and Weckx, 2016; Illeghems et al., 2015; Ho et al., 2014). Cocoa fermentation requires a sufficient depth of fermenting mass to ensure enough heat is generated during the process and it depends on which technique has being applied. Besides, fermenting mass should be covered properly to prevent heat from released to the environment and the container must have sufficient perforation for good drainage during sweating.
In addition, the fermenting mass should be turned at least once throughout the duration of the fermentation process to allow good aeration and ensure beans are mixed uniformly (Amoa-Awua, 2014; Ahmad Kamil et al., 2013).
2.2.1(c) Drying
Drying is a continuation process which carried out as soon as fermentation is completed. Completion of the process is manifested by decreasing temperature of the fermenting mass but there is no exact timer to stop the process.
Usually, the process is terminated based on the farmer experience (Saltini et al., 2013;
Nielsen, 2006). The aims of drying process are to reduce the moisture content of the wet cocoa beans from 40-60% to only 7.5%. This will ensure a good storage condition for dried cocoa beans and prevent the growth of moulds. The low moisture
22
content can deactivate the endogenous enzymes and prevent the over-fermented or off-flavour beans due to excessive proteolysis reaction. Besides, drying also ensures that colour development in cocoa beans takes place, changing the reddish colour to fully brown. The changes of colour are reported to correlate with reduction of the bitter and astringent taste of dried cocoa beans (Saltini et al., 2013; Thompson et al., 2013; Zahouli et al., 2010).
Normally, the drying process is performed naturally under the sun by spreading the beans on the appropriate surface, preferably on an elevated platform.
The process will take about three to seven days depending on the weather as well as the thickness of cocoa beans layer. The cocoa bean layer is limited to ‘one bean thickness' or 5 cm especially on the first day for optimum penetration of sunlight during drying. It there is some mistake in this process, it will result in blackish beans due to the moulds. The cocoa beans need to be turned periodically (every two to three hours) to ensure all the beans are evenly warmed. Upon drying, cocoa beans are physical inspected either sufficiently dried or not by grabbing a handful of the beans and rubbed each other. The cocoa beans are sufficiently dried if crackle sound is produced. Moreover, artificial drying which using oil or solid fuels as a source of power may be resorted in rainy periods (Lee et al., 2014; Ahmad Kamil et al., 2013;
Musa, 2012; Hii et al., 2012; Hii et al., 2009).
2.2.2 Secondary Processing
At the secondary stage, the fermented dried cocoa beans will be converted into semi-finished product. Traditionally, the fermented dried cocoa beans are processed by the chocolate manufacturer which normally located in a temperate climate country. But nowadays, the cocoa growing countries are preferred to produce
23
cocoa liquor from their own beans. Regardless where the process is carried out, the principle of cocoa bean processing in this stage has not changed for more than 150 years, where the beans are still cleaned, de-shelled, roasted, and sometimes alkalized.
Subsequently, the beans will be ground into cocoa liquor before following two distinctive processing lines. Approximately 65% of the cocoa liquor is reported to be pressed into butter and cake before the cake will finally be pulverized into powder.
Whereas, the remaining 35% is processed into commercial cocoa liquor which for directly used by the manufacture of chocolate (Beckett, 2015b; De Zaan, 2009).
2.2.2(a) Beans cleaning
Cleaning step is performed to ensure various types of foreign material which may be left or mixed up during primary processing are removed. The foreign materials; especially plant debris should be removed because it may release unpleasant gasses during roasting which would be spoiled the cocoa aroma. Hard materials such as sand, stones and metals may damage the machinery, especially during grinding. The procedure starts with the fermented dried cocoa beans will be move through a coarse and fine sieve equipped with a vibratory to remove sand and stone while segregating the beans according to similar size within each batch. At the same time, a strong flow of air will act as suction which will draw off the dust, leaves and fibres, whereas iron and other metals will be removed by a magnet. Finally, the cleaned beans will go on to the next stage of processing (Beckett, 2015b; De Zaan, 2009). A few cocoa manufacturers adopt bean blending technique before cleaning procedure to minimize fluctuating characteristics of cocoa flavour.
24 2.2.2(b) Roasting
Fermented dried cocoa beans are reported to have a bitter, acidic, astringent and musty but rich with flavour and aroma precursors. Roasting will transform the fermentation products such as free amino acids, oligopeptides and reducing sugars via Maillard reactions into full characteristics of chocolate flavours as well as browning of the cocoa seeds (Sacchetti et al., 2015; Jinap, 2004). Unlike reactions during fermentation or drying, the Maillard reaction that is occurred during the roasting process is non-enzymatic and driven by thermal treatment as high as 150 °C. Therefore, roasting will virtually sterilize the beans from an excess of bacteria, fungi and moulds involved in fermentation that passed through drying (Beckett, 2015b;
Nazaruddin et al., 2006). In addition, roasting will further reduce the moisture content from about 7.5% to the ranged between 1.5 - 3% and also volatile acids such as acetic acid by evaporation (Beckett, 2015b).
Nowadays, there are three different kinds of roasting techniques for different purposes or products, namely whole bean, nib and liquor roasting (Ziegleder, 2009). The traditional roasting of the whole bean helps separate the seed coats (shell) from the cotyledons and makes cracking and winnowing much easier. However, the method will result in melting of the cocoa butter and subsequently migrates into the shell. When the shell is removed, up to 0.5% of cocoa butter is estimated to lost.
Another disadvantage of whole bean roasting is that there is always a range of different sized beans involved. This will lead to a heterogeneous level of roasting treatment and may result in poorer flavour as the larger beans being undercooked while the smaller beans are being over-roasted (Beckett, 2015b; Owusu et al., 2013). Alternatively, some chocolate manufacturers practise nib or cocoa liquor roasting where the beans are de-shelled, crushed and winnowed before roasting. The difference between liquor
25
roasting and nib roasting is where the nib will be further ground into a paste then liquor after winnowed. The advantages of practising nib or liquor roasting are reported as a more uniform distribution of heat, rapid evaporation of water from the nib and increase in output for the same amount of energy input (Beckett, 2015b;
Ziegleder, 2009).
2.2.2(c) Grinding
After roasting, winnowing which is the process to remove shells from the nib takes place. After that, the fermented, dried and roasted cocoa nibs are ground into cocoa liquor. The main objective of grinding is to make the cocoa particles small enough so that as much fat as possible is removed from the cells within the cotyledons. An additional reason is to make the nib readily for chocolate making process without necessary to further mill the nib (Beckett, 2015b). Later, the cocoa liquor will be pressed by hydraulic press into cocoa cake to extract cocoa butter. The cake is then pulverized into fine cocoa powder (Afoakwa, 2014).
2.2.3 Cocoa-Based Product Development
Cocoa is synonymous with chocolate but there is a wide range of product that can be developed from cocoa either by using husk, shell, powder, pulp or butter. In food and beverage, by-products of liquefied pulp such as gin, brandy, vinegar, wine, jam and pectin are developed as an extra income (Cudjoe et al., 2009). The cocoa pulp is processed into juice for refreshing drinks and ice cream (Chin, 2016). In cosmetic and personal care products such as toothpaste, mouth rinse, foundation, anti-wrinkle cream, scented perfume, lip balm, lipstick and soap from cocoa-based has been
26
developed (Azila et al., 2016; Akoto et al., 2015; Norliza, 2010; Azila and Nur Azilah, 2012; Yap and Aminah, 2011).
2.3 Biochemical Changes During Fermentation
The biochemical reaction of cocoa fermentation can be divided into two stages, namely as pulp and cotyledons fermentation.
2.3.1 Pulp Fermentation
The pulp fermentation is also known as external fermentation which involving activity of microorganisms on the pulp. Before the cocoa seeds are exposed to the environment, the pulp has a relatively low pH (3.0 - 4.0) as well as low oxygen tension that mainly due to the content of 0.5 - 2% citric acid and thickness of the pulp. This condition as well as high sugar content including pectin and saccharides, provide an excellent medium for growth of microorganisms which exist from the environment through either soil, air, dust, banana and plantain leaves as well as gunny used to cover fermenting mass, the utensils and equipment used, the fruit fly, workers hands or husk (De Vuyst and Weckx, 2016; Teng-Sing et al., 2016; Hamdouche et al., 2015: Crafack et al., 2013; Meersman et al., 2013). The yeast population flourishes the flora on the pulp for about first 24 to 48 hours of fermentation. During this phase, a typical alcoholic fermentation occurs with the yeasts metabolizing pulp sugars and citric acid to ethanol, carbon dioxide, glycerol, acetic acid, succinic acid, and heat. The resulting heat is enough to cause the increasing of mass temperature from an ambient temperature between 25 - 30 °C to 35 - 40 °C within 48 hours (De Vuyst and Weckx, 2016; Ho et al., 2014;
Papalexandratou et al., 2013).
27
At the same time, the pulp cell is broken down by the action of pectolytic enzymes which produced by certain yeasts result in liquefying of pulp. The liquefied pulp will drain as other called as “sweating” and carried the flakes of the pulp away.
The drainage reduces pulp thickness and forming spaces that in turn allows some air to percolate through the mass thus make the conditions become aerobic (De Vuyst and Weckx, 2016; Crafack et al., 2013; Daniel et al., 2009; Schwan and Wheals, 2004; Ardhana and Fleet 2003). The new pulp conditions make the environment become more convenient for lactic acid bacteria (LAB) to grow and become as coexistence with yeast during the process in between 24 to 72 hours of fermentation.
The LAB use citric acid as a co-substrate and convert it into lactic acid during heterolactate fermentation. Conversion of citric acid into lactic acid resultant slightly increases in pH of the pulp as well as changing the composition of the fermenting mass.
Consequently, this condition influences the microbial succession and in which favour the acetic acid bacteria (AAB) to growth. The AAB which produce acetic acid by oxidation of ethanol, are dominantly growth between 48 to 112 hours of fermentation (De Vuyst and Weckx, 2016; Ho et al., 2014; Papalexandratou et al., 2013).
As the fermentation has progressed, the concentration of acetic acid becomes higher especially after turning of the fermenting mass, where the aeration getting better and encourages the exothermic oxidation of ethanol to acetic acids.
Hence, liberates more heat as well as rising of mass temperature up to 45 - 50 °C (De Vuyst and Weckx, 2016; Moens et al., 2014). The rising of temperature beyond 45 °C is unfavourable not only to the acetic acid bacteria hence resulting in a decline of all microorganisms except for spore-forming aerophilic bacteria types or bacilli.
Starting from that moment onward, the bacilli comprise over 80% of microflora and dominate the mass environment. At this phase of fermentation, the pulp layer is
28
totally depleted hence the fermenting mass becomes more aerobic and together with water formation as well as good aeration promotes the temperature decrease.
Decreased of temperature is suggested as an indicator for fermentation to be ended (Lima et al., 2015; Schwan and Wheals, 2004). The biochemical changes occur during pulp fermentation is summarized in Figure 2.10.
Figure 2.10: Trend of microbial and metabolites changes during the pulp fermentation. (a) Microbial succession (Source: Kouame et al., 2015) and (b) metabolite changes (Source: De Vuyst and Weckx, 2016).
Counts Log CFU/gChanges of metabolites
29 2.3.2 Cotyledon Fermentation
Enzymatic and other biochemical reactions occur inside the cocoa beans during fermentation are known as cotyledon or internal fermentation (Lima et al., 2011). It starts with the diffusion of ethanol into the micropyle, changes the cellular structural causing loss of the of testa selective permeability. Consequently, this will allow the diffusion of acetic acid into the cotyledons, kill the embryo and will cause the death situation of beans (Anderson et al., 2006; de Brito et al., 2001). As the cocoa beans death, the membrane barrier has lost its function and allowed the cellular components such as seed enzymes (e.g. invertase, endoprotease, glycosidase and polyphenol oxidase) and substrates (e.g. anthocyanins, flavanols, phenols and storage proteins) are free to mix. The accumulation of acetic acid as well as other organic acids in the cotyledon will cause nib acidification, which in response to heat will be activated the cocoa enzymes. Hence, these will induce the onset of flavours precursors formation during fermentation (Brillouet and Hue, 2017; Kumari et al., 2016; Sousa et al., 2016; Kadow et al., 2015; Lima et al. 2011).
As previously mention, cocoa is a rich source of polyphenols which encompassed about 4% of anthocyanins, 37% of flavanols and 58% of proanthocyanidins. Anthocyanins are water-soluble pigment which consists of four compounds namely, cyanidin arabinoside, cyanidin galactoside, cyanidin rutinoside as well as cyanidin pentoside and are responsible for the colour of cacao seed. The pigment has the ability to convert orange-red to blue-violet colour in food and beverage products (Aprotosoaie et al., 2016; Bordiga et al., 2015; Voigt and Lieberei, 2014; Wallace and Giusti, 2011; Cakirer et al., 2010). Although in intact condition, the pigment has not given any marked taste or aroma, it is proven that there is an inverse relationship between flavour development and the retained
